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gAgTgC-3′ and exon 11 5′-gCCCGCAggTCCTCTTTCCC. TCACA-3′ of the housekeeping gene phosphoglucomutase-1. (PGM1) (GenBank Accession No.

EXPERIMENTAL IMMUNOLOGY doi: 10.1111/sji.12058 ..................................................................................................................................................................

Tumour Regression Induced by Co-administration of MIP-3a and CpG in an Experimental Model of Colon Carcinoma S. Arab*, M. Mojarrad†, M. Motamedi‡, R. Mirzaei*, M. H. Modarressi§ & J. Hadjati*

Abstract *Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran; †Department of Medical Genetics, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran; ‡Lorestan University of Medical Sciences, Khoramabad, Iran; and §Department of Human Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

Received 11 January 2013; Accepted in revised form 25 March 2013

CCL20/macrophage inflammatory protein-3a (MIP-3a) represents one of the potent chemoattractive proteins for dendritic cells (DCs). Herein, we investigated whether in vivo genetic modification of tumour cells aimed at intratumoural production of MIP-3a might lead to accumulation of DCs in tumour tissue. Mice injected with CT26, received recombinant adenovirus (Ad) vectors (AdMIP-3a) expressing MIP-3a protein. This was complemented by injections of CpG. Interestingly, MIP-3a gene therapy combined with CpG injections resulted in specific cytotoxicity. This was associated with significant suppression of tumour growth rate. These findings demonstrate the potential of strategies that utilize in vivo overexpression of chemokines.

Correspondence to: J. Hadjati, Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Poursina Avenue, Keshavarz Blvd, Tehran 1417613151, Iran. E-mail: [email protected]

Introduction Dendritic cells (DCs) are sentinels of the immune system that instruct the activation of T and B lymphocytes to initiate immune responses [1]. Based on the major role of DCs in starting immune responses, a range of approaches have been established to use DCs for promoting immune response against tumour antigens. These approaches include pulsing DCs with tumour-associated antigens or apoptotic tumour cells or changing DCs genetically, using RNA derived from tumour cells, genes coding for tumourassociated antigens [2–6]. DCs are rare in the peripheral blood and generating DCs from peripheral blood mononuclear cells (PBMC) is both time consuming and pricey. These matters have restricted the use of ex-vivo-generated DCs in the treatment of tumours [7–9]. CCL20/macrophage inflammatory protein-3a (MIP-3a) is a CC chemokine, which is a potent chemoattractant for DCs both in humans and mice [10–12]. This chemokine has been shown to attract DCs in vitro and in vivo and plays a critical role in the directional migration of DCs to peripheral tissues [13–17]. Thus, it seems reasonable that induced expression and elevated concentration of CCL20 in tumour tissues might work as an approach to prompt

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antitumour immunity, through attracting a high number of DCs to the tumour site. To avoid manipulation of DCs ex-vivo, we hypothesized that if tumours could be genetically altered in vivo to produce MIP-3a, the consequence should be the migration and accumulation of DCs within the tumour area. The newly infiltrated DCs could then be matured using intratumoural injection of CpG oligonucleotides. Herein, we evaluated this hypothesis in a murine model of colon carcinoma. A recombinant adenoviral vector expressing MIP-3a protein was used to transfer and express the chemokine inside the tumour. Injections of CpG-containing oligonucleotides were performed around the tumour site to stimulate DC maturation. This approach may be able to elicit a strong antitumour immune response against tumour cells and assist to eradicate them.

Materials and methods Animals and cell line. Six- to eight-week-old female BALB/c mice were purchased from Laboratory Animal Center, Institute Pasteur of Iran. Mice were handled and tested according to the local guidelines for animal care and ethics. BALB/c-derived colon carcinoma (CT26) and fibrosarcoma

Ó 2013 John Wiley & Sons Ltd.

S. Arab et al. Injection MIP-3a and CpG to Activate Antitumour Immune Responses 29 ..................................................................................................................................................................

(WEHI164) cell lines were obtained and cultured in RPMI 1640 (Sigma, Steinheim, Germany), supplemented with 10% heat-inactivated foetal bovine serum (FBS) (Gibco, Grand Island, MA, USA), 2 mM L-glutamine (Sigma), 100 lg/ml streptomycin and 100 U/ml penicillin. Recombinant adenovirus production. AdMIP-3a and AdNull recombinant adenoviruses were produced as previously described [18]. AdMIP-3a and AdNull were replication-deficient serotype 5-based adenovirus vectors with E1 and E3 deletions in which the mouse MIP-3a cDNA and no transgene, respectively, are under transcriptional control of the cytomegalovirus immediate-early enhancer and promoter. The recombinant viruses were amplified, purified using centrifugation, ultrafiltration and tittered as described previously [19–21]. Oligonucleotides. The oligonucleotides used in this study included CpG ODN 1826 (3′-TCC ATG ACG TTC CTG ACG TT-5′) and the non-CpG control (CpGc) ODN (3′TCC AGG ACT TTC CTC AGG TT-5′). Both of the purified ODNs were purchased from Alpha DNA Company (Montreal, QC, Canada). Generation of bone marrow–derived dendritic cells. Bone marrow–derived DCs (BMDCs) were generated as described by Inaba et al. [22] with slight modifications. Briefly, bone marrow cells were obtained from the femur and tibia of female BALB/c mice. Following washing with PBS, cells (1 9 106 cells/ml) were placed in 24-well plates in RPMI plus 10% FBS, 20 ng/ml GM-CSF (R&D Systems, Gothenburg, Sweden) and 10 ng/ml IL-4 (R&D Systems). After 3 days, non-adherent cells were gently collected and fresh media were added. Non-adherent DCs with the typical morphologic features of dendritic cells were harvested at day 7 of the culture and were used for the in vitro migration assay to test the function of the recombinant AdMIP-3a vector. Reverse transcription-polymerase chain reaction (RT-PCR). To confirm the expression of MIP-3a in HEK 293T (Human Embryonic Kidney) cells infected with recombinant adenovirus, total RNA of infected cells was extracted after 48 h, using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. cDNA was synthesized using M-MuLV reverse transcriptase and random hexamers in the presence of RNasin (MBI-Fermentas, St LeonRot Germany). cDNA quality was checked using primers designed from exon 10 5′-TCCgACTgAGCggCACTgggAgTgC-3′ and exon 11 5′-gCCCGCAggTCCTCTTTCCC TCACA-3′ of the housekeeping gene phosphoglucomutase-1 (PGM1) (GenBank Accession No. NM_002633). The PCR amplification was carried out in a 25-ll reaction mixture containing 1x PCR buffer (50 mM KCL, 50 mM Tris-HCl, pH = 8.4 and 1.5 mM of MgCl2), each dNTP at a concentration of 200 lM, each primer at a concentration of 0.4 lM, and 1 unit of Taq polymerase enzyme. 1 ll cDNA was added to reaction mixture. Reaction tubes were placed in Eppendorf thermal cycler (Eppendorf AG, Hamburg, Germany) programmed for 30

Ó 2013 John Wiley & Sons Ltd.

cycles of denaturation at 94 °C for 45 s, 45 s of annealing at 64 °C and 50 s of extension at 72°°C; an additional 3 min of denaturation at 94 °C preceded the first cycle and a final extension step of 7 min. 10 ll of PCR products was run on 2% agarose gel (Invitrogen, London, UK), stained with ethidium bromide (EB), and then photographed under ultraviolet light. Positive samples were selected for MIP-3a amplification. A pair of primers (forward—MIPf: 5′-ATggCCT gCggTggCAAgC-3′, reverse MIPr: 5′TTACATCTTCTTg ACTCTTAggC-3′) were designed to amplify a 294-bp coding region of MIP-3a (GenBank Accession No: NM_016960). Amplification was performed in the same reaction mixture as described above except for MIP-3aspecific primers. PCR reactions consisted of preliminary denaturation at 95 °C for 3 min followed by 35 cycles of 95 °C for 30s, 55 °C for 30s and 72 °C for 45 s followed by a 7-min final extension at 72 °C. 10 ll of PCR products was run on 2% agarose gel (Invitrogen), stained with ethidium bromide, and then photographed under ultraviolet light. Identity of PCR product band was confirmed by enzymatic digestion using either HindIII or PstI restriction enzymes, separately. Digestion reactions contained 5 ll PCR product, 4 units enzyme, 1 ll 109 digestion buffer and H2O to a final volume of 10 ll. Reactions were allowed to proceed for 2 h at 37 °C. Protein expression assay. To evaluate the MIP-3a protein generated by the recombinant AdMIP-3a-infected cells, 3 days after infection, supernatants from infected cells and cell lysates were harvested and examined by Western blotting. Briefly, cell 29 sample buffer (50 mM Tris-HCl pH6.8, 100 mM DTT, 2%w/v SDS, 0.1% bromophenol blue, 10% glycerol) was added to either equal volume of supernatant or cell plate. Mixtures were heated in 95 °C for 5 min and then centrifuged in 10,000 g for 3 min. Mixtures were incubated on ice until electrophoresis. Total protein was electrophoresed through 10% denaturing SDSPAGE using 200 V for 2 h, then electrotransferred onto nitrocellulose membrane (Millipore) by 100v overnight. Membrane was blocked in PBS containing 5% BSA for 2 h at room temperature (RT). Rat anti-mouse MIP-3a (R&D Systems Inc., Minneapolis, MN, USA) was used as the primary antibody. After three times washing with PBS, membrane was incubated with HRP-linked Ig secondary antibody with shaking at RT for 2 h. Finally, C1N4 chromogen substrate was used for visualization of MIP-3a band based on manufacturer’s instruction (Amersham Life Sciences Inc., Arlington Heights, IL, USA). In vitro chemotaxis assay. Cell migration assay was carried out in 24-well micro chemotaxis chamber (Corning Costar Corporation, Cambridge, MA, USA) as described previously [23]. Briefly, dendritic cells were suspended at a concentration of 106 cells/ml in RPMI 1640 medium containing 2 mM glutamine and 10% FBS, and applied to the top wells of the chamber. The bottom chamber contained 600 ll of

30 Injection MIP-3a and CpG to Activate Antitumour Immune Responses S. Arab et al. .................................................................................................................................................................. supernatant of HEK293T cells transfected with recombinant AdMIP-3a or AdNull or recombinant mouse MIP-3a (rmMIP-3a) (R&D Systems Inc.). After 3 h incubation at 37 °C, migrated cells were separated from bottom chamber and stained with PE-conjugated mAbs against CD11c (BD PharMingen, San Diego, CA, USA) and counted with a FACS Analysis System (Becton–Dickinson, Oxford, UK). Tumour challenge and immunotherapy. Tumour cells (2 9 105 CT26 in 100 ll PBS) were injected subcutaneously in the right flank of mice. Seven days later, AdMIP-3a or AdNull in 100 ll PBS (2 9 108 pfu) or PBS alone (100 ll) was administered intratumourally. At days 9, 11 and 13, CpG or CpGc (25 lg) was injected around the tumour site. When the tumour was palpable, the shortest and longest surface diameters were measured every 2 days using digital calipers. The tumour area was achieved by multiplying the shortest and longest axis of the tumour, and then the mean tumour growth rate per 48 h was calculated. Cytotoxicity assay. Three weeks after tumour challenge, splenocytes were isolated and used as effector cells. Tumour cell lines (CT26, WEHI164) were used as target cells. Cytotoxic activity was measured by lactate dehydrogenase (LDH) cytotoxicity detection kit (Roche Applied Science, Mannheim, Germany). After washing the effector and target cells with assay medium (RPMI1640 with 1% BSA), the effector cells were cocultured with target cells in a 96well round bottom plate for 6 h at 37 °C. The plates were then centrifuged, and the supernatants were transferred to another flat-bottom ELISA plate. One hundred microlitre of LDH detection mixture was added to each well and incubated for 30 min. Absorbance was measured by an ELISA reader at 490 nm. The percentage of cell-mediated cytotoxicity was determined by the following equation:

Cytotoxicity ð%Þ ¼

HEK293T cells cDNA (Fig. 1A). To confirm the identity of the amplified fragment, we digested purified PCR products with HindIII and PstI. According to Webcutter 2.0 online software analysis results [24], HindIII and PstI cleave the desired PCR product at nucleotides 170 and 67, respectively (Fig. 1B). Digestion of our PCR products generated the same fragments, confirming the specificity of the amplification. Western blot analysis using an antimouse MIP-3a monoclonal antibody was performed to confirm protein expression. MIP-3a was easily detected in both the cell lysate and the culture supernatant of transfected HEK293T cells (data not shown). Chemoattractant activity of MIP-3a

The biological function of expressed MIP-3a protein was tested in a chemotaxis assay, where the chemotactic activity of conditioned medium from HEK293T cells transfected with AdMIP-3a or AdNull was examined. The maximal chemotactic activity was observed with recMIP-3a (Fig.2). The media from recMIP-3a and AdMIP-3a transfected cells were chemotactic, and the AdMIP-3a appeared to be at least two folds more effective than the AdNull. These results indicated that HEK293T cells infected with AdMIP-3a could secrete biologically active soluble MIP3a into the cell culture supernatant. Tumour-Specific CTL responses induced by intratumoural administration of AdMIP-3a

To determine whether MIP-3a gene therapy combined with CpG injections can stimulate a CTL response, splenocytes were isolated from mice three weeks after tumour challenge

ðexperimental release  spontaneous target release  spontaneous effector releaseÞ ðmaximal target release  spontaneous target releaseÞ  100%:

Statistical analysis. The results were expressed as mean  SD. Statistical analysis was performed using a one-way ANOVA. P-value

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